Mechanisms involving in the onset of arteriosclerosis have been gradually elucidated in these days, and risk factors thereof have been identified. Especially, hypercholesterolemia, hypertension, diabetes, and smoking are recognized to be manifest four risk factors, thus the therapeutic treatments have been extensively carried out. Clinically common pathologies of these disease states are insulin resistance. The meaning of insulin resistance is nearly equivalent to the reduction of sensitivity to insulin in cells, thereby the actions of insulin upon the uptake of sugar into the cells are deteriorated. The insulin resistance may be caused due to the abnormalities in secretion of insulin itself, abnormalities of insulin receptors on target cells, abnormalities of an intracellular signaling system, and reduced supply of sugar to the tissue based on peripheral circulatory disorder that is caused hemodynamically. Reaven, 1988, reports that many pathological states are developed due to the insulin resistance, and designates a pathological state as “syndrome X” that may concurrently represent insulin resistance, glucose tolerance abnormalities, hyperinsulinemia, hypertriglyceridemia, hypo-HDL cholesterolemia and hypertension, and further suggests that the pathological state syndrome X closely participates in the onset of arteriosclerosis (Reaven, G. M. et al., Diabetes, 37, 1595-1607, 1988).
In addition, sugar supply to the cells is known to be generally decreased through insulin resistance, accompanied by enhancement of insulin secretion from pancreas, thus leading to hyperinsulinemia. Therefore, several problems in connection with insulin resistance have been raised in clinical fields. For example, insulin resistance and hyperinsulinemia are reported to promote-diabetic nephritis (Niskanen, L. et al., Diabetes, 41, 736-741, 1993), and to elevate frequency of diabetic retinopathy (Yip, J. et al., Lancet, 341, 369-370, 1993). Moreover, insulin resistance has been reported to enhance plasminogen activator inhibitor 1 (PAI-1), to deteriorate the functions of a blood fibrinolytic system (Potter van Loon B J et al., Metab. Clin. Exp., 42, 945-954, 1993), and to trigger arterial atherosclerosis (Sato, Y. et al., Diabetes, 38, 91-96, 1989).
Prevalence rate of diabetes accounts for 5% of the total population, and approximately six millions of Japanese citizens are suffering from diabetes. Diabetes comprises insulin dependent diabetic mellitus (IDDM) and insulin independent diabetic mellitus (NIDDM). Reportedly, IDDM accounts for about 7% of the total diabetes cases, whilst NIDDM does about 90%. In particular, a significant causative factor of the onset of NIDDM that corresponds to a majority of diabetes has been conceived as the insulin resistance.
To date, tyrosine phosphorylation has been elucidated to play important roles in signal transduction of insulin. Insulin receptor is a hetero-tetramer of two glycoprotein subunits, namely an α-subunit having a molecular weight of 135 kDa and a β-subunit having a molecular weight of 95 kDa, which are bound through disulfide bonds resulting in α2β2 structure. The α-subunit has an insulin binding activity, while the β-subunit has a protein tyrosine kinase (PTK) domain that is activated by autophosphorylation. Accordingly, when insulin is bound to the α-chain of an insulin receptor, certain tyrosine residues existing in the β-chain of the insulin receptor are autophosphorylated. The activity of insulin receptor tyrosine kinase is further promoted through the tyrosine autophosphorylation. It is reported that thus activated insulin receptor tyrosine kinase phosphorylates tyrosine residues of IRS (insulin receptor substrate), the intracellular substrates thereof, and signal transduction is proceeded through recognition and binding of the tyrosine-phosphorylated insulin receptors by Ash/Grb2 or PI-3 kinase, finally resulting in manifestation of biological activities of insulin, such as glucose uptake, sugar metabolism and cell proliferation (see, FIG. 9, Goldstein, B. J. et al., Receptor, 3, 1-15, 1993; and Kanai, F. et al. Biochemical and Biophysical Research Communications, 195(2), 762-768, 1993). In this signal transduction pathway, however, an enzyme tyrosine phosphatase, which inactivates the activated insulin receptors, i.e., protein tyrosine phosphatase (hereinafter referred to as PTP), has not been progressively studied.
The serious studies of PTPs were initiated after completion of cloning of PTP1B gene and elucidation of the nucleotide sequence thereof by Fischer et al. in 1988, which is cytoplasmic PTP derived from human placenta (Tonks, N. K. et al., J. Biol. Chem., 263, 6722-6730, 1988; Charbonneau, H. et al., Proc. Natl. Acad. Sci. USA, 85, 7182-7186, 1988). Consequently, homology to PTP1B could be observed not with the known serine/threonine phosphatases but with two cytoplasmic regions of CD45, a transmembranous molecule involved in a hemopoietic system. Moreover, CD45 was also revealed to have PTP activity (Tonks, N. K. et al., Biochemistry, 27, 8695-8701, 1988; and Charbonneau, H. et al., Proc. Natl. Acad. Sci. USA, 85, 7182-7186, 1988).
To date, many PTPs have been cloned based on their homologies of cDNA sequences, and new PTPs have been reported subsequently (Streuli, M. et al., J. Exp. Med., 168, 1523-1530, 1988; Krueger, N. X. et al., EMBO J., 9, 3241-3252, 1990; and Trowbridge, I. S. et. al., Biochim. Biophys. Acta, 1095, 46-56, 1991). PTPs can be classified generally to: (1) membrane type PTPs having transmembrane region (LCA, leukocyte common antigen, namely CD45, as well as LAR and PTP α, β, γ, δ, ε and ζ), and cytoplasm type PTPs without transmembrane region (PTP1B, TC-PTP, PTP-MEG, PTPH1, STEP and PTP1C).
Many of membrane type PTPs have two PTP homologous domains inside the cell (domain 1 and domain 2, see, FIGS. 1(a) and (b)). A sequence comprising cysteine (signature motif), Ile/Val-His-Cys-Xaa-Ala-Gly-Xaa-Xaa-Arg-Ser/Thr-Gly (SEQ ID NO: 2), has been conserved in the phosphatase domains between the PTPs reported heretofore. The research on crystallography of PTP1B revealed that the region forms small pockets on the surface of a PTP molecule, and that the cysteine residue is located to the bottom of the pocket, participating directly in binding of the molecule to phosphate (Barford, D. et al., Science, 263, 1397-1404, 1994). In addition, it was also revealed that the depth of the pocket of PTP may determine the specificity of serine/threonine phosphatase because phosphate that is binding to serine or threonine cannot reach to the pocket of the enzymatic active center of PTP1B. Moreover, the importance of the above-mentioned signature motif in exhibiting the enzymatic activity has been elucidated (Streuli, M. et al., EMBO J., 9, 2399-2407, 1990). Taking into account of these observations, it has been conceived that the conserved cysteine in domain 1 may play an important role in exhibiting the enzymatic activity, and domain 2 may determine the substrate specificity of the enzyme.
Among a group of PTPs, LAR derived from human is a prototype of receptor type protein tyrosine phosphatases, which was cloned from human placental genome library using a phosphatase domain of CD45, a receptor type protein tyrosine phosphatase, as a probe (Streuli M. et al., J. Exp. Med., 168, 1553-1562, 1988). CD45 is specifically expressed on hemocytic cells, whilst LAR is expressed on the cells other than hemocytic cells, particularly in insulin sensitive organs such as liver and skeletal muscle (Goldstein B. J., Receptor, 3, 1-15, 1993). LAR is especially interesting among many types of receptor type PTPs due to its similarity of the extracellular domain with cell adhesion molecules. The entire structure of LAR is elucidated as having 150 kDa of extracellular E-subunit that consists of Ig-like domains and fibronectin type III domains, and 85 kDa of P-subunit comprising a transmembrane region and an intracellular domain having two phosphatase domains, which are covalently bound immediately outside of the cell membrane (see, FIG. 1, Streuli M. et al, EMBO J., 11, 897-907, 1992).
A large number of functional roles of LAR have been reported to date. For example, it was reported that: responses to neurotrophin are decreased in LAR deficient nerves (Yang, T. et al., 27th Annual Meeting of the Society for Neuroscience, New Orleans, La., USA, Oct. 25-30, 1997, Society for Neuroscience Abstracts, 23, 1-2, 1997); secretion of apolipoprotein B is decreased by suppression of LAR activity (Phung, T. L. et al., Biochemical and Biophysical Research Communications, 237(2), 367-371, 1997); and loss of expression of LAR diminishes the size of cholinergic nerve cells of prosencephalon basement, thus control by the cholinergic nerve cells at hippocampal dentate gyrus is deteriorated (Yeo, T. T. et al., J. Neurosci. Res., 47(3), 348-360, 1997). In such a manner, it has been gradually revealed that LAR is bearing several important roles in a living body. Furthermore at present, the most remarkable researches are directed to the relationships between LAR and insulin receptors (Hashimoto, N. et al., J. Biol. Chem., 267(20), 13811-13814, 1992).
In 1995, a literature was presented which should be noted, reporting that LAR tyrosine phosphatase activity is abnormally increased in adipose tissue of an obese person, with such an increase being suggested as a cause of onset of insulin resistance and a risk factor of cardiovascular diseases (Ahmad, F. et al., J. Clin. Invest., 95(6) 2806-2812, 1995). Several reports followed thereafter illustrating that LAR is closely concerned with insulin receptors (Mooney, R. A. et al., Biochemical and Biophysical Research Communications, 235(3), 709-712, 1997; Orr, S. R et al., Biochemical Society, Transaction, 25(3), 452S, 1997; Ahmad, F. et al., J. Clin. Investigation, 100(2), 449-458, 1997; Ahmad, F. et al., J. Biol. Chem., 272(1), 448-457, 1997; Norris, K. el al., Febs Letters, 415(3), 243-248, 1997; and Li, P. M. et al., Cellular Signaling, 8(7), 467-473, 1996). Further, on the basis of such information, Ahmad, F. et al. recently reported that PTP1B may be a therapeutic target of disease states involving in insulin resistance (Ahmad, F. el at, Metabolism, Clinical and Experimental, 46(10), 1140-1145, 1997). From the researches to date on PTPs such as LAR, CD45 and the like, it has been elucidated that PTPs bear extremely important roles in an intracellular signaling system.
In 1992, Streuli et al. reported that binding between LAR E-subunit and P-subunit may be dissociated due to the noncovalency of their binding, and thus E-subunit is specifically shed from the cell membrane surface (Streuli, M. et al. EMBO J., 11(3), 897-907, 1992). However, because many researchers have focused the studies using polyclonal or monoclonal antibodies elicited against a LAR E-subunit that is an extracellular domain thereof, a P-subunit even solely having phosphatase activities has been ignored. For example, when an anti-LAR antibody is used intending measurement of LAR phosphatase assay, total phosphatase activity could not be measured unless an antibody to P-subunit is employed. In view of such circumstances, the present inventors started to produce antibodies that specifically bind to a LAR P-subunit, particularly to an intracellular domain thereof, without any specificity to CD45.
Known antibodies to protein tyrosine phosphatase include an antibody generated using 196 amino acid residues as an antigen spanning from the transmembrane region of CD45 to a part of phosphatase domain 1 (Transduction Laboratories). However, it is unclear how these antibodies are immunospecific to phosphatase domains of LAR and the other protein tyrosine phosphatases. Therefore, it was also necessary to produce antibodies which are specific to a LAR intracellular domain but not to CD45.
Thyroid tumors include benign adenoma and malignant carcinoma. At present, palpation, ultrasonic diagnosis, fine needle aspiration cytology, and diagnosis on tissue sections are clinically carried out in order to diagnose thyroid tumors. Thyroid tumors can be classified into adenoma, papillary carcinoma, follicular carcinoma, undifferentiated (anaplastic) carcinoma, medullary carcinoma and malignant lymphoma, whilst thyroid carcinoma (papillary and follicular cancers) can be generally classified into differentiated and poorly differentiated types.
On diagnosis of thyroid carcinoma, if abnormalities were found on palpation and ultrasonic diagnosis, cytological examination with fine needle aspiration has been predominantly carried out because of fewer burdens to the patient, and in difficult cases where definite diagnosis is impossible, additional histological diagnosis is carried out in which thyroid tissue is excised. However, such histological diagnosis imposes more burdens to the patient, and there exist possibilities to excise together with normal tissue. In fact, discrimination by cytological examination is often difficult to draw exact diagnosis, thus many cases have been nevertheless entrusted to histological examination. Additionally, fine needle aspiration cytology does not result in definite diagnosis because cell-cell bindings may be destroyed in those specimens compared to the morphologic observation on tissue sections. Furthermore, in almost cases of follicular carcinoma, discrimination between benign and malignant tumors can be difficult even though histological diagnosis is performed as well as cytological examination. Accordingly, it has been strongly desired by clinicians or pathologists to develop new tools that can discriminate benign/malignant tumors in fine needle aspiration cytology even in such difficult cases for diagnosis as in follicular carcinoma.